Marker for prenatal diagnosis and monitoring

ABSTRACT

The present invention relates to new methods for diagnosing a pregnancy-associated disorder by analyzing fetal DNA present in the mother&#39;s blood. More specifically, this invention relies on the discovery that the maspin gene is differentially methylated in fetal DNA and in maternal DNA and provides these new diagnostic methods, which distinguish fetal DNA from maternal DNA and detect prenatal disorders based on abnormalities in fetal DNA level and methylation status.

This application is a continuation of U.S. patent application Ser. No.15/012,611, filed Feb. 1, 2016, now U.S. Pat. No. 9,862,999, which is acontinuation of U.S. patent application Ser. No. 13/215,024, filed Aug.22, 2011, now abandoned, which is a division of U.S. patent applicationSer. No. 12/724,335, filed Mar. 15, 2010, now U.S. Pat. No. 8,026,067,which is a division of U.S. patent application Ser. No. 11/144,951,filed Jun. 3, 2005, now U.S. Pat. No. 7,709,194, which claims priorityto U.S. provisional patent application No. 60/577,242, filed Jun. 4,2004, the contents of each of the above are herein incorporated byreference in the entirety.

BACKGROUND OF THE INVENTION

Early detection of pregnancy-related conditions, including potentialcomplications during pregnancy or delivery and genetic defects of thefetus is of crucial importance, as it allows early medical interventionnecessary for the safety of both the mother and the fetus. Prenataldiagnosis has been routinely conducted using cells isolated from thefetus through procedures such as chorionic villus sampling (CVS) oramniocentesis. These conventional methods are, however, invasive andpresent an appreciable risk to both the mother and the fetus despitemost careful handling (Tabor et al., Lancet 1:1287-1293, 1986).

Alternatives to these invasive approaches have been developed forprenatal screening, e.g., to detecting fetal abnormalities, followingthe discoveries that several types of fetal cells can be found inmaternal circulation (Johansen et al., Prenat. Diagn. 15:921-931, 1995)and more importantly, circulating cell-free fetal DNA can be detected inmaternal plasma and serum (Lo et al., Lancet 350:485-487, 1997). Theamount of fetal DNA in maternal blood has been shown to be sufficientfor genetic analysis without complex treatment of the plasma or serum,in contrast to the necessary steps for isolating and enriching fetalcells. Fetal rhesus D (RhD) genotyping (Lo et al., N. Engl. J. Med.339:1734-1738, 1998), fetal sex determination (Costa et al., N. Engl. J.Med. 346:1502, 2002), and diagnosis of several fetal disorders (Amicucciet al., Clin. Chem. 46:301-302, 2000; Saito et al., Lancet 356:1170,2000; and Chiu et al., Lancet 360:998-1000, 2002) have since beenachieved by detecting fetal DNA in maternal blood using a polymerasechain reaction (PCR)-based technique.

In addition, quantitative abnormalities of fetal DNA in maternalplasma/serum have been reported in preeclampsia (Lo et al., Clin. Chem.45:184-188, 1999 and Zhong et al., Am. J. Obstet. Gynecol. 184:414-419,2001), fetal trisomy 21 (Lo et al., Clin. Chem. 45:1747-1751, 1999 andZhong et al. Prenat. Diagn. 20:795-798, 2000) and hyperemesis gravidarum(Sekizawa et al., Clin. Chem. 47:2164-2165, 2001). Detection of fetalnucleic acid in maternal blood for prenatal genetic analysis is alsodisclosed in U.S. Pat. No. 6,258,540.

Fetal RNA present in maternal blood has also been established as adiagnostic tool for pregnancy-associated conditions. For instance, U.S.patent application Ser. No. 09/876,005 discloses non-invasive techniquesbased on detection of fetal RNA in maternal blood; U.S. patentapplication Ser. No. 10/759,783 further discloses that the amount ofcertain mRNA species (e.g., hCG-β, hCRH, hPL, KISS1, TPFI2, and PLAC1)present in maternal blood can be used as markers for diagnosing,monitoring, or predicting pregnancy-related disorders such aspreeclampsia, fetal chromosomal aneuploidy, and preterm labor.

Although the stability of DNA provides an advantage for fetal DNA-baseddiagnosis, one major limitation does exist for this approach: both fetaland maternal DNA is present in the acellular portion of a pregnantwoman's blood, e.g., serum or plasma. Thus, there is a need todistinguish fetal DNA from maternal DNA to ensure accurate diagnosis. Itwas first disclosed in U.S. patent application Ser. No. 09/944,951,published as 20030044388, that fetal and maternal DNA may bedistinguished by their different methylation profile. Landes et al. inU.S. Patent Application Publication No. 20030211522 also proposeddifferential methylation markers may be used for prenatal diagnosis. Inthe present disclosure, one particular gene, the mammary serine proteaseinhibitor (maspin) gene, is identified for the first time as a genecontaining regions differentially methylated in genomic DNA originatedfrom a fetus or from an adult (e.g., a pregnant women) due to thedifferent status of gene expression. Thus, the differentially methylatedfetal maspin gene allows proper identification or quantification offetal and maternal DNA and therefore reliable diagnosis of prenatalconditions.

BRIEF SUMMARY OF THE INVENTION

In one aspect, this invention relates to a method for detecting ormonitoring a pregnancy-associated disorder in a woman pregnant with afetus. This method comprises the following steps: (a) obtaining a bloodsample from the woman; (b) determining the methylation status of atleast a portion of the maspin gene in the blood sample, wherein theportion of the maspin gene from the fetus and the portion from the womanare differentially methylated, thereby distinguishing the maspin genefrom the woman and the maspin gene from the fetus in the blood sample;(c) determining the level of the fetal maspin gene; and (d) comparingthe level of the fetal maspin gene with a standard control. In somecases, an increase from the standard control indicates the presence orprogression of a pregnancy-associated disorder. In other cases, adecrease from the standard control indicates the presence or progressionof a pregnancy-associated disorder.

In some embodiments, the blood sample is whole blood. In otherembodiments, the blood sample is plasma or serum. In an exemplaryembodiment, the portion of the maspin gene from the woman is methylatedand the portion from the maspin gene from the fetus is less methylated.In another exemplary embodiment, step (b) is performed by treating theDNA present in the blood sample with a reagent that differentiallymodifies methylated and non-methylated DNA. An often-used reagent fordifferential modification of methylated and non-methylated DNA isbisulfite. Other suitable regents may include one or more enzymes thatpreferentially cleave either methylated or unmethylated DNA.Pregnancy-associated disorders can be detected or monitored by thismethod include preeclampsia, preterm labor, hyperemesis gravidarum,ectopic pregnancy, fetal chromosomal aneuploidy (such as trisomy 18, 21,or 13), and intrauterine growth retardation.

In another aspect, this invention provides a method for detecting ormonitoring a pregnancy-associated disorder in a woman pregnant with afetus. The method comprises the following steps: (a) obtaining DNA in ablood sample from the woman; (b) treating the DNA from step (a) withbisulfite; (c) performing an amplification reaction using the DNA fromstep (b) and two primers to amplify at least a portion of the maspingene, wherein the portion of the maspin gene from the fetal DNA and theportion of the maspin gene from the maternal DNA in the blood sample aredifferentially methylated, and wherein at least one of the two primersbinds differentially to the portion of the maspin gene from the fetus;and (d) comparing the level of the amplified portion of the maspin genefrom step (c) with a standard control. In some cases, an increase fromthe standard control indicates the presence or progression of apregnancy-associated disorder. In other cases, a decrease from thestandard control indicates the presence or progression of apregnancy-associated disorder.

In some embodiments, the blood sample is whole blood. In otherembodiments, the blood sample is plasma or serum. Some exemplaryamplification reactions include polymerase chain reaction (PCR), nucleicacid sequence based amplification, strand displacement reaction, andbranched DNA amplification reaction. Pregnancy-associated disorders canbe detected or monitored by this method include preeclampsia, pretermlabor, hyperemesis gravidarum, ectopic pregnancy, fetal chromosomalaneuploidy (such as trisomy 18, 21, or 13), and intrauterine growthretardation.

In a further aspect, this inventions relates to a method for detectingthe maspin gene from a fetus in the blood of a pregnant woman. Themethod comprises the following steps: (a) obtaining a blood sample fromthe woman; and (b) detecting at least a portion of the maspin gene,wherein the portion of the maspin gene is differentially methylated fromthe portion of the maspin gene from the maternal DNA in the bloodsample, thereby detecting the maspin gene from the fetus. In someembodiments, the blood sample is whole blood. In other embodiments, theblood sample is plasma or serum.

In a yet further aspect, the invention relates to a method for detectingand monitoring a pregnancy-associated disorder. This method comprisesthe following steps: (a) obtaining DNA in a blood sample from the woman;(b) treating the DNA from step (a) with a reagent that differentiallymodifies methylated and non-methylated DNA; (c) determining thenucleotide sequence of at least a portion of the maspin gene from step(b); and (d) comparing the profile of the nucleotide sequences from step(c) with a standard control, wherein a change in the profile from thestandard control indicates the presence or progression of apregnancy-associated disorder.

In some embodiments, the reagent comprises bisulfite. Or the reagent mayinclude one or more enzymes that preferentially cleave DNA when the DNAis either methylated or unmethylated. In other embodiments, the bloodsample is plasma or serum. In other embodiments, the method furthercomprises an amplification step of using the DNA from step (b) and twoprimers to amplify a portion of the maspin gene, wherein the portion ofthe maspin gene from the fetal DNA and the portion from the maternal DNAin the blood sample are differentially methylated, and wherein at leastone of the two primers binds differentially to the portion of the maspingene from the fetus. In an exemplary embodiment, the amplification stepis performed by polymerase chain reaction (PCR) or methylation-specificPCR; in another exemplary embodiment, step (c) is performed by massspectrometry, primer extension, polynucleotide hybridization, real-timePCR, or electrophoresis.

In an additional aspect, this invention relates to a method fordetecting trisomy 18 in a fetus in a pregnant woman. This methodcomprises the following steps: (a) obtaining DNA from a blood samplefrom the woman; (b) treating the DNA from step (a) with a reagent thatdifferentially modifies methylated and non-methylated DNA; and (c)determining the levels of different alleles of the maspin gene from thefetal DNA, thereby determining the ratio of the alleles, wherein thedifferent alleles have different methylation profile in at least portionof the maspin gene, and wherein an increase or a decrease in the ratiofrom a standard control indicates the presence of trisomy 18 in thefetus.

In some embodiments, the reagent comprises bisulfite. Or the reagent mayinclude one or more enzymes that preferentially cleave DNA when the DNAis either methylated or unmethylated. In other embodiments, the bloodsample is plasma or serum. In yet other embodiments, placental tissuesor other fetal tissues may be used for comparison. In other embodiments,the method further comprises an amplification step of using the DNA fromstep (b) to amplify of at least a portion of the maspin gene that isdifferentially methylated in the maspin gene from the fetal DNA and themaspin gene from the maternal DNA in the blood sample. In an exemplaryembodiment, the amplification step is performed by PCR ormethylation-specific PCR; in another exemplary embodiment, step (c) isperformed by mass spectrometry, primer extension, polynucleotidehybridization, real-time PCR, or electrophoresis.

Furthermore, this invention relates to a method for detecting trisomy 18in a fetus carried by a pregnant woman. The method includes thefollowing steps: (a) obtaining a blood sample from the woman; (b)determining the methylation status of at least a portion of the maspingene in the blood sample, wherein the portion of the maspin gene fromthe fetus and the portion from the woman are differentially methylated,thereby distinguishing the maspin gene from the woman and the maspingene from the fetus in the blood sample; and (c) determining the levelsof two different alleles of the fetal maspin gene, wherein a deviationof the ratio of the levels of the two alleles from 1:1 indicates trisomy18 in the fetus. In some embodiments of this method, the two differentalleles of the fetal maspin gene comprise a single nucleotidepolymorphism (SNP). One exemplary SNP is located at 156 bp upstream fromthe transcription start site of the maspin gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of the maspin genomic sequence,including the promoter and exon 1. The position of 2 CpG islands areshown as solid black bars. Arrows marked “F” and “R” denote the locationof the bisulfite sequencing primers used in our study. The genomic andexon sequences are derived from NT_025028 and NM_002639 (GenBankaccession numbers). CpG sites are shown in small vertical barsunderneath.

FIG. 2. Maspin promoter sequence after complete bisulfite conversion.The boxed sequence represents a fully methylated state at all CpG sites,which are numbered with respect to the transcription start site (+1).Bisulfite sequencing primers F and R are underlined with >>>and<<<,respectively. Methylation-specific PCR (MSP) primers are shownunderneath. MF and MR are used for MMSP, which detects the methylatedsequence, while UF and UR are for UMSP, which detects the unmethylatedsequence. Artificial mismatches, shown in lowercase, were added to the3^(rd) base from the 3′ end of the primers to enhance specificity andsensitivity of the MSP assays. MP and UP are the TaqMan MGB (Minor GroveBinding) probes designed for the real-time quantitative MMSP and UMSPassays, respectively.

FIGS. 3A-3B. Percentage of methylation of cytosine residues in CpG sitesof the maspin promoter. Data are shown for the paired placental tissueand maternal buffy coat from 8 first trimester pregnancies (FIG. 3A),and 8 third trimester pregnancies (FIG. 3B). The lines inside the boxesdenote the medians. The boxes mark the interval between the 25th and75th percentiles.

FIG. 4. Schematic diagram depicting the design for the U-maspinMassEXTEND reaction. The location of the −156 SNP is indicated in squarebrackets. The A/C SNP when bisulfite converted and interrogated in thereverse sense becomes a T/A SNP. The nested primers for the U-maspinMassEXTEND reaction (line arrows) are indicated schematically.Nucleotide positions on the extension primer which correspond to theunmethylated CpG sites are indicated by the positions marked “A”(adenine). hME: homogenous MassEXTEND.

FIG. 5. Schematic diagram for detecting fetal trisomy 18 in maternalplasma. Closed and open circles represent methylated and unmethylatedcytosines in CpG sites of the maspin promoter. The figures of 95% and 5%are merely for illustrative purposes only and represent one of thepossible relative concentrations of fetal and maternal DNA in maternalplasma. Arrows represent UMSP primers that amplify only the unmethylatedmaspin (U-maspin) sequences of the fetus, but not the methylated maspinsequence of the mother. Hence, the allelic ratio of any singlenucleotide polymorphism (SNP) within this region, in this case C/A,could be determined by analyzing the U-maspin PCR product. If this SNPis heterozygous for an euploid fetus, the allelic ratio will be 1:1.However, for a fetus with trisomy 18, the allelic ratio will deviatesignificantly from normal, and in one scenario may be 2:1. This U-maspinassay can thus be used for the non-invasive diagnosis of trisomy 18 bymolecular analysis of the maternal plasma.

FIG. 6A-6D. Methylation status of CpG sites in the maspin promoter.Juxtaposed are data from placental tissues (FIG. 6A) and correspondingmaternal buffy coat (FIG. 6B) from each of eight pregnancies in thefirst trimester (TP11, TP12, TP13, TP14, TP15, TP16, TP17, and TP21) andthe same tissues (FIGS. 6C and 6D) from each of eight pregnancies in thethird trimester (NP23, NP27, NP28, NP29, NP30, NP31, NP32, and NP40).Open and closed circles represent unmethylated and methylated cytosineresidues, respectively. At least 9 randomly chosen clones, numbered inthe column, were sequenced for each site of each tissue.

FIG. 7. Box plots of U-maspin concentrations in first-, second- andthird-trimester maternal plasma. Line within each box denotes themedian. Limits of the box denote the 25^(th) and 75^(th) percentiles.Whiskers denote the 5^(th) and 95^(th) percentiles. Filled circlesdepict the outliers.

FIG. 8. U-maspin concentration in maternal plasma before and 24 hoursafter delivery. Paired samples from the same pregnancy are depicted byidentical symbols connected by a line.

FIG. 9. Correlation between U-maspin and SRY concentrations in maternalplasma.

FIG. 10. U-maspin concentrations in maternal plasma of women withpreeclamptic (PET) and healthy (Normal) pregnancies. Line within eachbox denotes the median. Limits of the box denote the 25^(th) and 75^(th)percentiles. Whiskers denote the 5^(th) and 95^(th) percentiles. Filledcircles depict the outliers.

FIG. 11. Mass spectrometric tracings of maternal plasma U-maspin −156SNP genotype in cases 310 and 454. The corresponding maternal blood celland placental tissue genotypes for the two cases are shown in Table 1.In both mass spectra, the x-axis depicts the molecular weight of thedetected extension products (shown as sharp peaks), while the y-axisdepicts the intensity in arbitrary units. The expected positions of theA- and C-alleles are as marked.

FIG. 12. Scatter plot of the ratios of the −156 SNP among placentalU-maspin sequences in normal pregnancies and pregnancies involving atrisomy 18 fetus (T18). The ratios are determined by comparing the areaof the peaks for each respective allele on the mass spectra.

FIG. 13. Mass spectrometric tracing illustrating the U-maspin −156 SNPallelic frequency in two sample mixtures containing 95% DNA frommaternal blood cells and 5% DNA from placental tissues obtained frompregnancies involving a karyotypically normal fetus. The x-axis depictsthe molecular weight of the detected extension products (shown as sharppeaks), while the y-axis depicts the intensity in arbitrary units. Theexpected positions of the A- and C-alleles are as marked.

DEFINITIONS

The term “pregnancy-associated disorder,” as used in this application,refers to any condition or disease that may affect a pregnant woman, thefetus the woman is carrying, or both the woman and the fetus. Such acondition or disease may manifest its symptoms during a limited timeperiod, e.g., during pregnancy or delivery, or may last the entire lifespan of the fetus following its birth. Some examples of apregnancy-associated disorder include ectopic pregnancy, preeclampsia,preterm labor, and fetal chromosomal abnormalities such as trisomy 13,18, or 21.

The term “epigenetic state” or “epigenetic status” as used herein refersto any structural feature at molecular level of a nucleic acid (e.g.,DNA or RNA) other than the primary nucleotide sequence. For instance,the epigenetic state of a genomic DNA may include its secondary ortertiary structure determined or influenced by, e.g., its methylationpattern or its association with cellular proteins.

The term “methylation profile” or “methylation status,” when used inthis application to describe the state of methylation of a gene, refersto the characteristics of a DNA fragment relevant to methylation. Suchcharacteristics include, but are not limited to, whether any of thecytosine (C) residues within this DNA sequence are methylated, locationof methylated C residue(s), percentage of methylated C at any particularstretch of residues, and allelic differences in methylation due to,e.g., difference in the origin of the alleles or the level of geneexpression.

The term “single nucleotide polymorphism” or “SNP” as used herein refersto the polynucleotide sequence variation present at a single nucleotideresidue within different alleles of the same gene, e.g., the maspingene. This variation may occur within the coding region or non-codingregion (i.e., in the promoter region) of a gene. Detection of one ormore SNP allows differentiation of different alleles of a single gene.

The term “blood” as used herein refers to a blood sample or preparationfrom a pregnant woman or a woman being tested for possible pregnancy.The term encompasses whole blood or any fractions of blood, such asserum and plasma as conventionally defined.

The term “bisulfite” as used herein encompasses all types of bisulfites,such as sodium bisulfite, that are capable of chemically converting acytosine (C) to a uracil (U) without chemically modifying a methylatedcytosine and therefore can be used to differentially modify a DNAsequence based on the methylation status of the DNA.

As used herein, a reagent that “differentially modifies” methylated ornon-methylated DNA encompasses any reagent that modifies methylatedand/or unmethylated DNA in a process through which distinguishableproducts result from methylated and non-methylated DNA, thereby allowingthe identification of the DNA methylation status. Such processes mayinclude, but are not limited to, chemical reactions (such as a C→Uconversion by bisulfite) and enzymatic treatment (such as cleavage by amethylation-dependent endonuclease). Thus, an enzyme that preferentiallycleaves methylated DNA is one capable of cleaving a DNA molecule at amuch higher efficiency when the DNA is methylated, whereas an enzymethat preferentially cleaves unmethylated DNA exhibits a significantlyhigher efficiency when the DNA is not methylated.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleicacids (DNA) or ribonucleic acids (RNA) and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogs of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, single nucleotide polymorphisms (SNPs), and complementarysequences as well as the sequence explicitly indicated. Specifically,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues (Batzer et al.,Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem.260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98(1994)). The term nucleic acid is used interchangeably with gene, cDNA,and mRNA encoded by a gene.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) involved in thetranscription/translation of the gene product and the regulation of thetranscription/translation, as well as intervening sequences (introns)between individual coding segments (exons).

In this application, the terms “polypeptide,” “peptide,” and “protein”are used interchangeably herein to refer to a polymer of amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical mimetic of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers and non-naturally occurring amino acid polymers. As usedherein, the terms encompass amino acid chains of any length, includingfull-length proteins (i.e., antigens), wherein the amino acid residuesare linked by covalent peptide bonds.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.

Amino acids may be referred to herein by either the commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

As used in this application, an “increase” or a “decrease” refers to adetectable positive or negative change in quantity from an establishedstandard control. An increase is a positive change preferably at least2-fold, more preferably at least 5-fold, and most preferably at least10-fold of the control value. Similarly, a decrease is a negative changepreferably at least 50%, more preferably at least 80%, and mostpreferably at least 90% of the control. Other terms indicatingquantitative changes or differences from a comparative basis, such as“less,” are used in this application in the same fashion as describedabove.

A “polynucleotide hybridization method” as used herein refers to amethod for detecting the presence and/or quantity of a polynucleotidebased on its ability to form Watson-Crick base-pairing, underappropriate hybridization conditions, with a polynucleotide probe of aknown sequence. Examples of such hybridization methods include Southernblotting and Northern blotting.

“Primers” as used herein refer to oligonucleotides that can be used inan amplification method, such as a polymerase chain reaction (PCR), toamplify a nucleotide sequence based on the polynucleotide sequencecorresponding to a gene of interest, e.g., the maspin gene in variousmethylation states. At least one of the PCR primers for amplification ofa polynucleotide sequence is sequence-specific for the sequence.

“Standard control” as used herein refers to a sample comprising a geneof a predetermined amount or methylation profile (which may includemultiple different and separable characteristics related to methylation)suitable for the use of a method of the present invention, in order forcomparing the amount or methylation status of a particular gene, e.g.,the maspin gene, that is present in a test sample. A sample serving as astandard control provides an average amount or methylation profile of agene of interest that is typical for a defined time (e.g., firsttrimester) during pregnancy in the blood of an average, healthy pregnantwoman carrying a normal fetus, both of who are not at risk of developingany pregnancy-associated disorders or complications.

The term “average,” as used in the context of describing a pregnantwoman who is healthy, carries a chromosomally normal fetus, does not andwill not develop any pregnancy-associated conditions (such as ectopicpregnancy, preeclampsia, or preterm labor), refers to certaincharacteristics, such as the methylation profile of a particular gene(e.g., the maspin gene) of both maternal and fetal origins found in thewoman's blood, that are representative of a randomly selected group ofhealthy women who are pregnant with chromosomally normal fetuses and notsusceptible to any pregnancy-related diseases or conditions. Thisselected group should comprise a sufficient number of women such thatthe average amount or methylation profile of the gene of interest amongthese women reflects, with reasonable accuracy, the correspondingprofile in the general population of healthy pregnant women with healthyfetuses. In addition, the selected group of women generally have asimilar gestational age to that of a woman whose blood is tested forindication of a potential pregnancy-associated disorder. The preferredgestational age for practicing the present invention may vary depends onthe disorder that is being screened for. For example, a pregnant womanis screened for the risk of preeclampsia preferably during the secondtrimester of the pregnancy, whereas fetal chromosomal aneuploidy ispreferably screened for and diagnosed as early as possible. Moreover,the preferred gestational age for testing may also depend on the gene ofinterest in testing.

The term “preeclampsia” as used herein refers to a condition that occursduring pregnancy, the main symptom of which is various forms of highblood pressure often accompanied by the presence of proteins in theurine and edema (swelling). Preeclampsia, sometimes called toxemia ofpregnancy, is related to a more serious disorder called “eclampsia,”which is preeclampsia together with seizures. These conditions usuallydevelop during the second half of pregnancy (after 20 weeks), thoughthey may develop shortly after birth or before 20 weeks of pregnancy.

The term “preterm labor” or “premature labor” as used herein refers tothe condition where labor that begins more than three weeks before thefull gestation period of about 40 weeks, which often leads to prematurebirth if not treated.

The term “hyperemesis gravidarum” refers to extreme, persistent nauseaand vomiting during pregnancy, particularly during the first trimester.The nausea and vomiting may lead to dehydration and prevent necessaryweight gain for the pregnancy.

An “ectopic pregnancy” refers to an abnormal pregnancy in which afertilized egg has implanted outside the uterus. Although in most casesof ectopic pregnancy the egg settles in the fallopian tubes, this termalso encompasses abnormal pregnancies where the fertilized egg isimplanted in a woman's ovary, abdomen, or cervix.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The presence of fetal DNA in maternal plasma was first reported in 1997and offers the possibility for non-invasive prenatal diagnosis simplythrough the analysis of a maternal blood sample (Lo et al., Lancet350:485-487, 1997). To date, numerous potential clinical applicationshave been developed. In particular, quantitative abnormalities of fetalDNA concentrations in maternal plasma have been found to be associatedwith a number of pregnancy-associated disorders, including preeclampsia,preterm labor, antepartum hemorrhage, invasive placentation, fetal Downsyndrome, and other fetal chromosomal aneuploidies. Hence, fetal DNAanalysis in maternal plasma has been suggested as a potential marker forthe monitoring of fetomaternal well-being.

However, fetal DNA co-exists with background maternal DNA in maternalplasma. Hence, most reported applications have relied on the detectionof Y-chromosome sequences as these are most conveniently distinguishablefrom maternal DNA. However, such an approach limits the applicability ofthe existing assays to only 50% of all pregnancies with male fetuses.Thus, there is much need for the development of gender-independent fetalDNA markers for maternal plasma detection.

It was previously demonstrated that fetal and maternal DNA can bedistinguished by their differences in methylation status (U.S. PatentApplication Publication No. 20030044388). Methylation is an epigeneticphenomenon, which refers to processes that alter a phenotype withoutinvolving changes in the DNA sequence. By exploiting the difference inthe DNA methylation status between the paternally- andmaternally-inherited alleles at H19, a locus exhibiting genomicimprinting (differential methylation and hence differential expressionof two alleles of a single gene, related to the parental origin of aparticular allele), one (Y.M.D. Lo) of the present inventors and hisgroup first demonstrated the feasibility of using epigenetic markers todetect fetal-derived maternally-inherited DNA sequence from maternalplasma (Poon et al., Clin. Chem. 48:35-41, 2002). Landes et al. havealso proposed the use of epigenetic markers for non-invasive prenataldiagnosis (U.S. Patent Application Publication No. 20030211522).

The present inventors have recently demonstrated that placenta-derivedRNA can be detected in maternal plasma (Ng et al., Proc. Natl. Acad.Sci. USA 100:4748-4753, 2003). On the other hand, it has been shown thatplasma DNA in normal individuals is predominantly derived fromhematopoietic cells (Lui et al., Clin. Chem. 48:421-427, 2002). Thus, ithas been hypothesized that the predominant source of maternal DNA isderived from peripheral blood cells while the placenta is a possiblesource of fetal DNA release into maternal plasma. Hence, one strategyfor the development of a generic fetal-specific DNA marker for detectionin maternal plasma is to identify a gene that is differentiallymethylated between the placenta and the maternal peripheral blood cells.

Maspin (mammary serine protease inhibitor) is a protein belonging to thefamily of serine protease inhibitors. It is found to be expressed in avariety of normal tissues, mainly those of epithelial origin, such asbreast, prostate, placenta, testis, colon and the small intestines. Theclinical significance of maspin was realized when a study by Zou et al.(Science 263:526-529, 1994) demonstrated its reduced expression in humanbreast carcinoma cells. Subsequent studies noted an inverse relationshipbetween its expression and cancer prognosis or the presence ofmetastasis. To date, the maspin gene is widely accepted as a tumorsuppressor gene, whose physiological function lies in the promotion ofcell-matrix adhesion and the inhibition of cell invasion. Investigationsfail to reveal DNA mutations that are responsible for the alteredexpression of maspin in tumors. Instead, maspin was found to be underepigenetic control where expression is suppressed by promotermethylation and histone deacetylation (Futscher et al., Nat. Genet.31:175-179, 2002; Maass et al., Biochem. Biophys. Res. Commun.297:125-128, 2002). Futscher et al., supra, observed that the 19 CpGsites spanning the maspin promoter were uniformly unmethylated inmaspin-positive cell types, while they were densely methylated in allmaspin-negative cell types.

The present inventors demonstrated, for the first time, that the maspingene is differentially methylated between the fetal DNA from the fetus(e.g., from the placenta) and the maternal DNA from the mother'speripheral blood cells. This discovery thus provides a new approach fordistinguishing fetal and maternal DNA and new methods for non-invasiveprenatal diagnosis.

II. General Methodology

Practicing this invention utilizes routine techniques in the field ofmolecular biology. Basic texts disclosing the general methods of use inthis invention include Sambrook and Russell, Molecular Cloning, ALaboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Protein sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized, e.g., according to the solid phase phosphoramidite triestermethod first described by Beaucage & Caruthers, Tetrahedron Lett. 22:1859-1862 (1981), using an automated synthesizer, as described in VanDevanter et. al., Nucleic Acids Res. 12: 6159-6168 (1984). Purificationof oligonucleotides is performed using any art-recognized strategy,e.g., native acrylamide gel electrophoresis or anion-exchange highperformance liquid chromatography (HPLC) as described in Pearson &Reanier, J. Chrom. 255: 137-149 (1983).

The sequence of the maspin gene, a polynucleotide encoding the Maspinpolypeptide, and synthetic oligonucleotides can be verified using, e.g.,the chain termination method for sequencing double-stranded templates ofWallace et al., Gene 16: 21-26 (1981).

III. Acquisition of Blood Samples and Extraction of DNA

The present invention relates to analyzing the epigenetic status offetal DNA found in maternal blood as a non-invasive means to detect thepresence and/or to monitor the progress of a pregnancy-associatedcondition or disorder. Thus, the first steps of practicing thisinvention are to obtain a blood sample from a pregnant woman and extractDNA from the sample.

A. Acquisition of Blood Samples

A blood sample is obtained from a pregnant woman at a gestational agesuitable for testing using a method of the present invention. Thesuitable gestational age may vary depending on the disorder tested, asdiscussed below. Collection of blood from a woman is performed inaccordance with the standard protocol hospitals or clinics generallyfollow. An appropriate amount of peripheral blood, e.g., typicallybetween 5-50 ml, is collected and maybe stored according to standardprocedure prior to further preparation.

B. Preparation of Blood Samples

The analysis of fetal DNA found in maternal blood according to thepresent invention may be performed using, e.g., the whole blood, serum,or plasma. The methods for preparing serum or plasma from maternal bloodare well known among those of skill in the art. For example, a pregnantwoman's blood can be placed in a tube containing EDTA or a specializedcommercial product such as Vacutainer SST (Becton Dickinson, FranklinLakes, N.J.) to prevent blood clotting, and plasma can then be obtainedfrom whole blood through centrifugation. On the other hand, serum may beobtained with or without centrifugation following blood clotting. Ifcentrifugation is used then it is typically, though not exclusively,conducted at an appropriate speed, e.g., 1,500-3,000×g. Plasma or serummay be subjected to additional centrifugation steps before beingtransferred to a fresh tube for DNA extraction.

In addition to the acellular portion of the whole blood, DNA may also berecovered from the cellular fraction, enriched in the buffy coatportion, which can be obtained following centrifugation of a whole bloodsample from the woman and removal of the plasma.

C. Extraction of DNA

There are numerous known methods for extracting DNA from a biologicalsample including blood. The general methods of DNA preparation (e.g.,described by Sambrook and Russell, Molecular Cloning: A LaboratoryManual 3d ed., 2001) can be followed; various commercially availablereagents or kits, such as QiaAmp DNA Mini Kit or QiaAmp DNA Blood MiniKit (Qiagen, Hilden, Germany), GenomicPrep™ Blood DNA Isolation Kit(Promega, Madison, Wis.), and GFX™ Genomic Blood DNA Purification Kit(Amersham, Piscataway, N.J.), may also be used to obtain DNA from ablood sample from a pregnant woman. Combinations of more than one ofthese methods may also be used.

IV. Methylation-Specific Chemical Modification of DNA

Upon being extracted from a blood sample of a pregnant woman, the DNA istreated with a reagent capable of chemically modifying DNA in amethylation differential manner, i.e., different and distinguishablechemical structures will result from a methylated cytosine (C) residueand an unmethylated C residue following the treatment. Typically, such areagent reacts with the unmethylated C residue(s) in a DNA molecule andconverts each unmethylated C residue to a uracil (U) residue, whereasthe methylated C residues remain unchanged. This C→U conversion allowsdetection and comparison of methylation status based on changes in theprimary sequence of the nucleic acid. An exemplary reagent suitable forthis purpose is bisulfite, such as sodium bisulfite. Methods for usingbisulfite for chemical modification of DNA are well known in the art(see, e.g., Herman et al., Proc. Natl. Acad. Sci. USA 93:9821-9826,1996) and will not be discussed in detail here.

As a skilled artisan will recognize, any other reagents that are unnamedhere but have the same property of chemically (or through any othermechanism) modifying methylated and unmethylated DNA differentially canbe used for practicing the present invention. For instance,methylation-specific modification of DNA may also be accomplished bymethylation-sensitive restriction enzymes, some of which typicallycleave an unmethylated DNA fragment but not a methylated DNA fragment,while others (e.g., methylation-dependent endonuclease McrBC) cleave DNAcontaining methylated cytosines but not unmethylated DNA. In addition, acombination of chemical modification and restriction enzyme treatment,e.g., combined bisulfite restriction analysis (COBRA), may be used forpracticing the present invention.

V. Polynucleotide Sequence Amplification and Determination

Following the chemical modification of DNA in a methylation-differentialmanner, the treated DNA is then subjected to sequence-based analysis,such that the maspin gene from the fetal DNA may be distinguished fromthe maspin gene from the maternal DNA, and that fetal maspin genemethylation profile may be determined and compared to a standardcontrol.

A. Amplification of Nucleotide Sequences

An amplification reaction is optional prior to the maspin gene sequenceanalysis after methylation specific modification. In some embodiments ofthis invention, the amplification is performed to preferentially amplifya portion of the maspin gene that has a particular methylation pattern,such that only the maspin gene from one particular source, e.g., fromthe placenta or other tissues of the fetus, is detected and analyzed.

A variety of polynucleotide amplification methods are well establishedand frequently used in research. For instance, the general methods ofpolymerase chain reaction (PCR) for polynucleotide sequenceamplification are well known in the art and are thus not described indetail herein. For a review of PCR methods, protocols, and principles indesigning primers, see, e.g., Innis, et al., PCR Protocols: A Guide toMethods and Applications, Academic Press, Inc. N.Y., 1990. PCR reagentsand protocols are also available from commercial vendors, such as RocheMolecular Systems.

PCR is most usually carried out as an automated process with athermostable enzyme. In this process, the temperature of the reactionmixture is cycled through a denaturing region, a primer annealingregion, and an extension reaction region automatically. Machinesspecifically adapted for this purpose are commercially available.

Although PCR amplification of a target polynucleotide sequence (e.g., aportion of the maspin gene where the fetal and maternal sequence isdifferentially methylated) is typically used in practicing the presentinvention, one of skill in the art will recognize that the amplificationof a maspin gene sequence found in a maternal blood sample may beaccomplished by any known method, such as ligase chain reaction (LCR),transcription-mediated amplification, and self-sustained sequencereplication or nucleic acid sequence-based amplification (NASBA), eachof which provides sufficient amplification. More recently developedbranched-DNA technology may also be used to qualitatively demonstratethe presence of a particular maspin gene sequence (which represents aparticular methylation pattern), or to quantitatively determine theamount of a particular maspin gene sequence (which represents aparticular methylation pattern) in the maternal blood. For a review ofbranched-DNA signal amplification for direct quantitation of nucleicacid sequences in clinical samples, see Nolte, Adv. Clin. Chem.33:201-235, 1998.

B. Determination of Polynucleotide Sequences

Techniques for polynucleotide sequence determination are also wellestablished and widely practiced in the relevant research field. Forinstance, the basic principles and general techniques for polynucleotidesequencing are described in various research reports and treatises onmolecular biology and recombinant genetics, such as Wallace et al.,supra; Sambrook and Russell, supra, and Ausubel et al., supra. DNAsequencing methods routinely practiced in research laboratories, eithermanual or automated, can be used for practicing the present invention.Additional means suitable for detecting changes (e.g., C→U) in apolynucleotide sequence for practicing the methods of the presentinvention include but are not limited to mass spectrometry, primerextension, polynucleotide hybridization, real-time PCR, andelectrophoresis.

VI. Establishing a Standard Control

In order to establish a standard control for practicing the method ofthis invention, a group of healthy pregnant women carrying healthyfetuses are first selected. These women are of similar gestational age,which is within the appropriate time period of pregnancy for screeningof conditions such as preeclampsia, fetal chromosomal aneuploidy, andpreterm labor using the methods of the present invention. Similarly, astandard control is established using samples from a group of healthynon-pregnant women.

The healthy status of the selected pregnant women and the fetuses theyare carrying are confirmed by well established, routinely employedmethods including but not limited to monitoring blood pressure of thewomen, recording the onset of labor, and conducting fetal geneticanalysis using CVS and amniocentesis.

Furthermore, the selected group of healthy pregnant women carryinghealthy fetuses must be of a reasonable size, such that the averageamount of fetal maspin gene in the maternal blood or the methylationprofile of at least a portion of the fetal maspin gene in the maternalblood obtained from the group can be reasonably regarded asrepresentative of the normal or average amount or methylation profileamong the general population of healthy women carrying healthy fetuses.Preferably, the selected group comprises at least 10 women.

A standard control for fetal maspin gene methylation profile may reflectmultiple different and separable aspects of the methylation status ofthis gene. For example, one aspect of a methylation profile is whetherthe C residue is methylated or not; another aspect is the number ofmethylated C bases within a particular region of the maspin gene; afurther aspect of the profile is the percentage(s) of methylated C atany given locations. Additional aspects of a methylation profile mayinclude, but are not limited to, the allelic difference in methylation,the ratio of differentially methylated alleles, and the like. Fetalmaspin gene methylation profile may also vary depending on the tissuetype, e.g., placental or other fetal tissue. Thus, separate standardcontrols may be established for different fetal tissues used in testing.

Once an average level or methylation profile is established for thefetal maspin gene present in the maternal blood based on the individualvalues found in each woman of the selected healthy control group, thisaverage or median or representative value or profile is considered astandard control. Any blood sample that contains a similar amount of thefetal maspin gene or a similar methylation profile of the fetal maspingene can thus be used as a standard control. Furthermore, a solutioncontaining maspin DNA in the average or median or representative amountor of the average or median or representative methylation profile canalso be artificially assembled and serve as a standard control. Inaddition, separate standard controls may also be established fordifferent aspects of the methylation profile of the maspin gene.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially similar results.

The objectives of this study are:

(1) To systematically compare the methylation status of the maspin genebetween placental tissue and maternal peripheral blood cells;

(2) To develop an epigenetic assay for specific detection andquantification of placenta-derived maspin DNA in maternal plasma.

(3) To study the quantitative profile of placenta-derived maspin DNAconcentration in maternal plasma during the course of normal pregnancy;and

(4) To study the presence of quantitative aberrations in theconcentration of placenta-derived maspin DNA in maternal plasma inpregnancy-associated conditions, such as, but not limited topreeclampsia.

(5) To develop a method for the detection of trisomy 18 in a fetus in apregnant woman by determining the ratio of different alleles ofplacenta-derived maspin.

Materials and Methods

1. To Systematically Compare the Methylation Status of the Maspin Genebetween Placental Tissues and Maternal Peripheral Blood Cells

To demonstrate that difference does exist between the methylation statusof the maspin gene in the placenta and maternal blood, the presentinventors performed bisulfite sequencing on DNA extracted from 8 pairedplacental tissue and maternal peripheral venous blood from the thirdtrimester (38-39 weeks, median 38.5 weeks, SD 0.52 weeks) of pregnancy.In addition, for the purpose of prenatal monitoring, it would bebeneficial if the marker is detectable as early as possible duringpregnancy. Hence, bisulfite sequencing was also performed on DNAextracted from 8 paired chorionic villus samples (CVS) and maternalblood from the first trimester (9-13 weeks, median 10.5 weeks, SD 1.22weeks) of pregnancy. To demonstrate that this difference in themethylation status of the maspin gene is independent of the fetal sex,the inventors included both male (TP12, TP14, TP15, TP17, NP27, NP28,NP29, NP30, NP31, NP40) and female fetuses (TP11, TP13, TP16, TP21,NP23, NP32) for this part of the study.

Sample collection and processing. The tissues of first and thirdtrimesters were obtained respectively from pregnant subjects attendingclinic for prenatal diagnosis by chorionic villus sampling and fromthose requiring delivery by elective cesarean section at term. Sixmilliliters of the blood samples were collected in EDTA tubes and werecentrifuged at 1600×g for 10 min at 4° C. Plasma was carefullytransferred into plain polypropylene tubes for re-centrifugation at16000×g for 10 min at 4° C., and stored in fresh plain tubes without anycell pellet. The buffy coat portion was obtained after careful removalof plasma and stored separately at −20° C. Chorionic villus biopsy andplacenta after delivery were rinsed in phosphate buffered saline andstored in plain polypropylene tubes at −80° C.

Bisulfite sequencing. DNA was extracted from the placental tissues andmaternal buffy coat by using the QiaAmp DNA Mini Kit and QiaAmp DNABlood Mini Kit (Qiagen, Hilden, Germany) respectively according to themanufacturer's instructions. For each sample, 1 μg DNA was subjected tobisulfite conversion, which converts unmethylated cytosine residues touracil but leaves methylated cytosine residues unchanged, by theCpGenome DNA Modification Kit (Intergen, Burlington, Mass.) according tomanufacturer's instructions. The bisulfite converted DNA was thensubjected to PCR amplification with primers F and R (FIGS. 1 and 2)flanking the CpG sites, which may contain methylated cytosine residues,in the converted maspin promoter. These primers were designed not tobind to any potentially methylated cytosine residues. These primers areshown by way of illustration and should not be seen to limit the rangeof primers which can be used for this purpose. Reagents supplied in theTaqMan PCR Core Reagents Kit (Applied Biosystems, Foster City, Calif.)were used. In a final reaction volume of 50 μl, 1× Buffer II, 4 mMMgCl₂, 160 μM of each dNTP, 300 nM of each primer, 3% dimethylsulfoxide(DMSO), and 3U TaqGold polymerase were mixed. The thermal profileconsisted of an initial denaturation step of 95° C. for 10 min followedby 40 cycles of 95° C. for 1 min, 60° C. for 1 min, 72° C. for 1 min,and a final extension of 72° C. for 10 min. To analyze methylationstatus at the resolution of a single molecule, the PCR product wasTA-cloned into a plasmid vector using the pGEM-T Easy Vector System(Promega, Madison, Wis.) and the inserts from at least 10 positiverecombinant clones were analyzed by cycle sequencing using the BigDyeTerminator Cycle Sequencing v1.1 kit (Applied Biosystems) as per themanufacturer's instructions. After purification by genCLEAN columns(Genetix), 8 μl of the samples were added to 12 μl of Hi-Di formamideand run on a 3100 DNA Analyzer (Applied Biosystems).

Data comparison and statistical analysis. A CpG site was scored asmethylated if the sequence was cytosine; scored as unmethylated if itwas occupied by a thymine residue (deoxy counterpart of uracil). Theproportion of methylated cytosine residue for each CpG site in each typeof tissues was determined for each pregnancy. The distribution ofmethylated and unmethylated cytosines were compared between theplacental tissues and maternal buffy coat for each CpG site (FIG. 3).Statistical analysis was performed using the Sigma Stat 3.0 software(SPSS).

2. To Develop an Epigenetic Assay for the Specific Detection andQuantification of Placenta-derived Maspin DNA in Maternal Plasma

Fetal DNA coexists with a background of maternal DNA in plasma ofpregnant women. As demonstrated previously, plasma DNA is predominantlyderived from blood cells in normal, non-pregnant subjects. Based onthis, it is hypothesized that maternal DNA in maternal plasma is alsopredominantly derived from maternal blood cells in which the maspinpromoter is densely methylated. On the other hand, another study hasshown that fetal RNA detected in maternal plasma is derived from theplacenta. In light of this finding, it is hypothesized that thecontribution of fetal DNA in maternal plasma may also originate from theplacenta and hence possess the same methylation status in the maspinpromoter as that of the placenta. To test whether the maspin promoter inplacenta may be unmethylated, which would be different from thebackground of methylated maspin promoter of the maternal origin, it wasexamined if this epigenetic difference would allow fetal-specificdetection of placenta-derived maspin DNA in maternal plasma. It wasexamined whether unmethylated maspin sequences would:

(a) be detectable in plasma of pregnant women;

(b) increase in concentration as pregnancy progresses; and

(c) decrease substantially in concentration in maternal plasma afterdelivery of the fetus.

Methylation-specific PCR primer design. Based on the methylation mapgenerated from study (1) above, primers that discriminate between theunmethylated and methylated versions of the maspin promoter weredesigned, based on the principles of methylation-specific PCR (MSP)(Herman et al., Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996). Theassays specific for the unmethylated maspin (U-maspin) and methylatedmaspin (M-maspin) promoters were designated as UMSP and MMSP,respectively. The primers for UMSP (UF and UR) and for MMSP (MF and MP)are shown in FIG. 2. We employed the double Amplification RefractoryMutation System approach, which involves the use of two allele-specificprimers simultaneously during PCR when there is a need to distinguish asequence of interest from two or more closely related sequences. Toenhance the specificity of the primers, an additional mismatch (shown aslower case in primer sequences of FIG. 2) was introduced into the thirdbase from the 3′ end of both primers.

Real-time quantitative methylation-specific PCR. Two dual labeledfluorescent TaqMan MGB probes, MP and UP were designed to adopt the UMSPand MMSP respectively into the real-time quantitative assays. Theirsequences are shown in FIG. 2. Calibration curves were prepared byserial dilutions of high performance liquid chromatography-purifiedsingle stranded synthetic DNA oligonucleotides (Genset Oligos,Singapore) specific for the respective amplicons, with concentrationsranging from 1×10⁷ copies to 5 copies. Quantitative MSP data wereexpressed as copies of unmethylated or methylated DNA per milliliter ofplasma. The detectability of the unmethylated maspin promoter sequence(U-maspin) in maternal plasma collected from normal pregnancies duringthe third trimester, just before elective cesarean section and afterdelivery of the fetus, and also during all three trimesters of pregnancywere determined using the UMSP quantitative assay. As blood cells havemethylated maspin promoter, it is expected that the MSP system formethylated maspin sequence (i.e., the MMSP system) would give a positiveresult in all plasma samples, whether from pregnant or non-pregnantindividuals. Thus, the MMSP assay in the real-time quantitative PCRformat was used as a positive control.

3. To Study the Quantitative Profile of Placenta-derived Maspin DNAConcentration in Maternal Plasma During the Course of Normal Pregnancy

The quantitative profile of U-maspin during normal pregnancy has beeninvestigated. Six milliliters of maternal blood from women in the first,second and third trimesters of pregnancies were collected forquantification. For the third trimester pregnancies, women who underwentelective cesarean section were recruited. Blood was collected beforedelivery and at 24 hours after delivery. The concentration of theU-maspin DNA was determined in these maternal plasma samples. SRYquantification (Lo et al., Am. J. Hum. Genet. 64:218-224, 1998) was alsoperformed for samples collected from male-carrying pregnancies.Correlation between the U-maspin and SRY quantitative data wasdetermined.

4. Placenta-derived Maspin DNA in Maternal Plasma and Serum as a Markerfor Pregnancy Complications

Quantitative aberrations of fetal DNA concentrations in maternal plasmaand serum have been associated with a number of pregnancy-associateddisorders. Placenta-derived maspin DNA can therefore be used as a markerfor predicting, detecting, diagnosing and monitoringpregnancy-associated disorders, including, but not limited topreeclampsia, preterm labor, hyperemesis gravidarum, ectopicpregnancies, molar pregnancies, intrauterine growth retardation andchromosomal aneuploidies, such as fetal Down syndrome, fetal trisomy 18,fetal trisomy 13. Maternal plasma U-maspin concentration was determinedby the real-time quantitative assay among 8 preeclamptic pregnant womenbearing fetuses of both sexes (median gestational age: 36.1 weeks) andfrom 16 gestational age matched pregnant women without preeclampsia ascontrols (median gestational age: 36 weeks).

5. Development of a Method for the Detection of Trisomy 18 in a Fetus ina Pregnant Woman by Determining the Ratio of Different Alleles ofPlacenta-derived Maspin.

Primer extension reaction for genotypic analysis of U-maspin. Genotypicanalysis of a single nucleotide polymorphism (SNP) positioned at 156 bpupstream of the transcription start site in the maspin promoter wasperformed on placental tissues, maternal blood cells and plasmacollected from eight fetomaternal pairs. SNP genotyping was performed ongenomic DNA from the placental tissues and maternal blood cells usingthe standard primer extension (homogenous MassEXTEND (hME)) protocol ona MassARRAY system (SEQUENOM, San Diego, Calif.), which is amatrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF)MS system (Tang et al., Proc. Natl. Acad. Sci. USA 96:10016-20, 1999).

A method for determining the −156 SNP genotype among the U-maspinsequences in maternal plasma was then developed. 0.8 mL of plasma wasbisulfite converted as described above. The maspin promoter wasamplified using nested primers, shown as hME primary and secondaryprimers in FIG. 4, by conventional PCR. MassEXTEND assay was performedaccording to manufacturer's instructions. The nested primers andextension primer were designed to anneal to U-maspin promoter sequences(FIG. 4). The extension reaction was performed using a terminator mixconsisted of ddCTP, ddGTP, ddTTP and dATP. The extension reaction beginsat the SNP site (FIG. 4).

Gene dosage assessment of fetal chromosome 18. Genotyping of the −156SNP was first performed on genomic DNA extracted from placental tissuescollected from both normal and pregnancies involving a fetus withtrisomy 18. Cases heterozygous for the −156 SNP were identified. DNAextracted from the placental tissues of these cases were bisulfiteconverted and U-maspin was amplified using the nested conventional PCRas described above. The MassEXTEND assay targeting the −156 U-maspin SNPalleles as described above was then applied to the PCR products. Theabundance of the two U-maspin alleles at the −156 SNP was determined bycomparing the respective peak areas for each allele as shown by the massspectra determined by the MassARRAY system. As maspin is located onchromosome 18 and U-maspin is derived from the placenta, the relativeabundance of the two U-maspin alleles is reflective of the number offetal chromosome 18 (FIG. 5). The reliability of the assay and allelicratio determination was assessed by testing two sample mixturescomprised of 95% DNA from a maternal blood cell sample and 5% DNA fromthe corresponding placental tissue obtained from a pregnancy involving akaryotypically normal fetus.

Results and Discussion

Comparison of Methyation Status of the Maspin Promoter Between PlacentalTissue and Maternal Peripheral Blood Cells

Bisulfite sequencing of paired tissues from the first and thirdtrimesters revealed that most of the CpG sites in the maspin promoter ofmaternal buffy coat DNA was predominately methylated, while that in theplacental tissue DNA was relatively unmethylated (FIG. 6A, 6B, 6C, and6D). For both trimesters, all of these eight CpG sites, located 194 to103 base pairs upstream of the transcriptional start, were demethylatedin the placental tissue, relative to the maternal buffy coat, to astatistically significant extent (Chi-square, P<0.0001; FIG. 3A and 3B).Hence, these results have verified the hypothesis that placental tissuethat expresses maspin indeed has an unmethylated promoter. Thisphenomenon is observed as early as the 10^(th) week of gestation andthus makes early detection of U-maspin possible.

There is no sign of genomic imprinting in the loci investigated for bothtype of tissues. However, in the placental tissue, some degree ofmicroheterogeneity was observed in the methylation profile. This couldbe attributed to the mixed cell types found in the placental tissuebeing used.

Specific Detection of Placenta-derived Maspin DNA in Maternal Plasma

As proven by the bisulfite sequencing data, an epigenetic differencedoes exist between the placental tissue and the maternal peripheralblood cells, and thus can be exploited to develop PCR assays forspecific detection. There are several criteria for choosing these pairsof primers. First, it is necessary to design a reasonably short (<120bp) amplicon, in order to optimize for sensitivity, especially whenbisulfite conversion destroys most of the DNA. Second, it is necessaryto have an amplicon long enough for placing the flourescent probe. MGBprobe was designed, since it requires a shorter probe DNA sequence. Inthe case of the present inventors, primers flanking CpG sites −170 and−147 are chosen, which give an amplicon of 89 base pairs for MMSP and 98base pairs for UMSP (FIG. 2).

U-maspin was detected in all the 8 samples of maternal plasma obtainedfrom third trimester and all the 15 samples of maternal plasma obtainedfrom third trimester, the 10 samples from second trimester and the 11samples from first trimester (FIG. 7). This demonstrated that U-maspincould be readily detected in pregnancies, as early as the 10^(th) weekof gestation, despite the placental tissue is only partiallydemethylated at this time.

After delivery of the fetus, the concentration of U-maspin in maternalplasma decreased almost to an undetectable level for all 6 pregnanciesrequiring cesarean section (FIG. 8). This result illustrated thatU-maspin in maternal plasma was predominantly derived from the fetus.Thus, U-maspin behaves similarly as the established fetal DNA marker,SRY (present on the Y chromosome), in maternal plasma, which also clearsafter delivery of the fetus.

U-maspin and bisulfite-converted SRY concentrations were positivelycorrelated (Pearson correlation, correlation coefficient, r=0.668 andP=0.02) in the plasma from 12 pregnant women bearing male fetuses (FIG.9).

Elevated Maternal Plasma U-maspin Concentration in Preeclampsia.

U-maspin DNA concentration was measured in the plasma obtained from 8preeclamptic pregnant women and 16 gestational age matched pregnantwomen without preeclampsia as controls. The median U-maspinconcentration in maternal plasma was 5.7-fold elevated in thepreeclamptic group (median 737.7 copies/mL, IQR 306.9-1397.0) relativeto the control group (median 130.3 copies/mL, IQR 110.7-286.2) (FIG.10). A statistically significant difference in the maternal plasmaU-maspin concentrations between the preeclamptic and control groups wasobserved (LogXact logistic regression by matched case-control analysis,P=0.01158).

Detection of Placenta-derived U-maspin in Maternal Plasma.

The −156 SNP is an A/C polymorphism. The fetal and maternal maspingenotypes, shown in Table 1, were determined from 8 third-trimesterpregnancies using genomic DNA collected from placental tissues and thecorresponding maternal blood cells. A MassEXTEND assay was designed tointerrogate the −156 SNP among U-maspin promoter sequences. The A-alleleof the −156 SNP would be extended by one base while the C-allele wouldbe extended by two bases (FIG. 4) which are resolved as peaks ofdifferent masses on MS (FIG. 11). Maternal plasma samples from the eightpregnancies were bisulfite converted and the U-maspin −156 SNP genotypewas assessed. The maternal plasma U-maspin genotypes were completelyconcordant with that of the placental tissues (Table 1). These dataconfirm that U-maspin in maternal plasma is derived from the placenta.

To demonstrate the specificity of the MassEXTEND assay towards U-maspin,bisulfite converted maternal blood cells, which comprised predominantlyof M-maspin, were also analyzed. No false-positive amplification fromany of the maternal blood cell samples was noted (data not shown). Massspectrometric tracings for maternal plasma U-maspin genotyping for tworepresentative cases are shown in FIG. 11.

TABLE 1 Genotype analysis of U-maspin in maternal plasma Maspinpromoter-156 SNP genotype Genomic DNA Bisulfite converted DNA CaseMaternal Fetal Maternal plasma U-maspin 258 A A A 272 AC AC AC 300 A ACAC 310 A AC AC 331 AC AC AC 340 AC A A 427 AC A A 454 AC A ANon-invasive Prenatal Diagnosis of Trisomy 18

The use of the U-maspin system for the non-invasive prenatal diagnosisof trisomy 18 is illustrated in FIG. 5. This strategy is possiblebecause the maspin gene is located on chromosome 18. One embodiment ofthis strategy is the use of primer sequences which amplify unmethylatedmaspin sequences from maternal plasma. As demonstrated above thesesequences are predominantly derived from the fetus. The primer sequencesare designed such that they encompass one or more polymorphisms. Bymeans of illustration, one possible type of polymorphism is the singlenucleotide polymorphism (SNP). As a further illustration of thisstrategy, if the fetus is heterozygous at the detected polymorphism,then the allelic ratio of the detected alleles can be measured, possiblyby primer extension and mass spectrometry, or by other methods known tothose skilled in the art. In one scenario, if the fetus has the normalsituation of two chromosome 18, then the allelic ratio will be 1 to 1.If the fetus has the abnormal situation of three chromosome 18 (i.e.,trisomy 18), then the allelic ratio will deviate from 1 to 1. As anotherillustration of this, the allelic ratio may become 2 to 1 or 1 to 2.

Gene dosage of U-maspin for 23 normal pregnancies and 2 pregnanciesinvolving a trisomy 18 fetus was determined using the MassEXTEND assaywhich targets the −156 SNP among unmethylated maspin sequences. Thefetuses of all these pregnancies are confirmed to be heterozygous forthe SNP based on genotype analysis on genomic DNA extracted fromplacental tissues. To assess the gene dosage of U-maspin,bisulfite-converted placental tissue DNA was assessed by the MassEXTENDassay. The SNP allelic ratio was determined by comparing the area of thepeaks for the respective alleles on the mass spectra. The ratio betweenthe two alleles among the trisomy 18 pregnancies deviated from that ofthe normal pregnancies (FIG. 12).

To demonstrate the reliability of the assay and the strategy for allelicratio determination in samples containing a minority fraction of fetalDNA with a high background of maternal DNA, the assay was applied to twosample mixtures comprised of 95% DNA from a maternal blood cell sampleand 5% DNA from the corresponding placental tissue. Both pregnancieswere confirmed to be karyotypically normal. The results are shown inFIG. 13. Case A represents a pregnancy involving a mother homozygous forthe A-allele and a heterozygous fetus at the maspin −156 SNP, and viceversa for case B. The lack of detection of the maternal C-allele forcase B confirms the fetal-specifcity of the assay. The A:C allelic ratiofor case A was 1.235 which is within the range of values obtained forkaryotypically normal pregnancies (FIG. 12). These data confirm theapplicability of the assay for allelic ratio determination in biologicalsamples containing fetal DNA within a high background of maternal DNA,one example being fetal DNA in maternal plasma.

All patents, patent applications, and other publications cited in thisapplication, including published amino acid or polynucleotide sequences,are incorporated by reference in the entirety for all purposes.

What is claimed is:
 1. A method of analyzing the maspin gene in theblood of a pregnant woman, comprising the steps of (a) treating DNAobtained from a blood sample taken from the woman with a reagent thatdifferentially modifies methylated and non-methylated DNA, therebydistinguishing methylated and unmethylated version of the maspin gene;and (b) performing an amplification reaction to amplify at least oneportion of the maspin gene.
 2. The method of claim 1, furthercomprising, prior to step (a), obtaining the blood sample from the womanand isolating DNA from the blood sample.
 3. The method of claim 1,wherein the DNA is acellular DNA in the blood sample.
 4. The method ofclaim 1, wherein the blood sample is whole blood.
 5. The method of claim1, wherein the blood sample is plasma or serum.
 6. The method of claim1, wherein the blood sample is obtained from the woman after 10 weeks ofgestation.
 7. The method of claim 1, wherein the reagent comprisesbisulfate.
 8. The method of claim 1, wherein the reagent comprises oneor more enzymes that preferentially cleave methylated DNA.
 9. The methodof claim 1, wherein the reagent comprises one or more enzymes thatpreferentially cleave unmethylated DNA.
 10. The method of claim 1,wherein the amplification reaction is a polymerase chain reaction (PCR).11. The method of claim 10, wherein the PCR is a methylation-specificPCR.
 12. The method of claim 10, wherein the PCR is real-time PCR. 13.The method of claim 1, wherein the amplification reaction is a nucleicacid sequence based amplification, a strand displacement reaction, or abranched DNA amplification reaction.
 14. The method of claim 1, furthercomprising, after step (b), sequencing the at least one portion of themaspin gene that has been amplified by the amplification reaction. 15.The method of claim 14, wherein step (a) the sequencing step comprisesmass spectrometry, primer extension, polynucleotide hybridization, orelectrophoresis.
 16. The method of claim 1, wherein the at least oneportion of the maspin gene is the promoter region of the maspin gene.